1. Technical Field
The present invention generally relates to stable bacterial strains that are genetically engineered to overproduce coronatine, as well as methods for optimizing the yield of coronatine from such strains. In particular, the invention provides APV1, a stable, genetically engineered strain of Pseudomonas syringae that overproduces coronatine at temperatures that are amenable to commercial production.
2. Background
Polyketides constitute a huge family of structurally diverse natural products including those with antibiotic, chemotherapeutic, and antiparasitic activities. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by gram-positive bacterial species including Streptomyces and other actinomycetes (Hopwood, 1997; Katz, 1997). However, Pseudomonas, a gram-negative bacterium, produces a variety of antimicrobial compounds from the polyketide pathway including coronatine, mupirocin (pseudomonic acid), pyoluteorin, and 2,4-diacetylphloroglucinol (Bender et al., 1999).
With respect to coronatine (COR), the molecule is composed of two building blocks of distinct biosynthetic origin (Parry et al., 1994). One of these is the ethylcyclopropyl amino acid coronamic acid (CMA), which is derived from L-isoleucine via the intermediacy of L-alloisoleucine (Parry et al., 1994). The other component is coronafacic acid (CFA), which is a polyketide derived from acetate, butyrate, and a four-carbon unit derived from α-ketoglutarate (Parry et al., 1994; Parry et al., 1996). A brief overview of the biosynthetic route to coronatine is shown in FIG. 1. With reference thereto, COR consists of the polyketide component CFA coupled via amide bond formation to the amino acid component, CMA. CFA is a polyketide derived from three units of acetate, and one unit each of pyruvate and butyrate. CMA is derived from isoleucine via alloisoleucine. CFA can also be coupled to L-alloisoleucine (aile) and L-isoleucine (ile) to form the coronafacoyl analogues, CFA-aile and CFA-ile, respectively.
In plants infected with coronatine-producing bacteria, the primary symptom elicited by the presence of coronatine is a diffuse chlorosis that can be induced on a wide variety of plant species (Bender et al., 1999). Coronatine is also known to induce hypertrophy (cell enlargement), inhibit root elongation, and stimulate ethylene production in plants (Kenyon et al., 1992; Sakai et al., 1979). Several research groups have noted the remarkable structural and functional homologies between coronatine and methyl jasmonate (MeJA), a plant growth regulator derived from the octadecanoid signaling pathway, which is elicited by biological stress (Feys et al., 1994; Weiler et al., 1994). Coronatine and MeJA induce analogous biological responses in many plant tissues, and several researchers have suggested that coronatine functions as a molecular mimic of the octadecanoid signaling molecules produced by higher plants (Feys et al., 1994; Weiler et al., 1994). However, coronatine does not function solely as a molecular mimic of MeJA in some plant species (Palmer and Bender, 1995), and the mechanism of action for coronatine remains unclear.
While coronatine may cause undesirable effects in plants, it has also been shown to have great practical value, both as an abscission agent (see, e.g., U.S. patent Publication No. 20030013609 to Burns & Bender), and as a compound to increase taxane production (see U.S. patent Publication No. 20030175913 to Steele et al.), both of which are herein incorporated by reference.
Overproduction of coronatine by strain Pseudomonas syringae DC3000-hrcC has been described (Penaloza-Vazquez et al., 2000). DC3000-hrcC is a strain that contains a transposon insertion in the hrcC gene, which encodes a component of the type III secretion system. While strain DC3000-hrcC does overproduce coronatine, due to the unstable nature of the transposon insertion, this strain has been deemed unsuitable for commercial production of COR.
The prior art has thus far failed to provide stable bacterial strains capable of producing large quantities of coronatine under conditions that are suitable for commercial production.